https://www.um.edu.mt/library/oar/handle/123456789/31625 2026-09-09T07:33:43Z https://www.um.edu.mt/library/oar/handle/123456789/148763 Title: Comprehensive profiling of lung cancer markers for early screening and detection Abstract: Lung cancer remains the leading cause of cancer-related mortality worldwide, largely due to late-stage diagnosis and the lack of effective non-invasive screening strategies. The stability of extracellular vesicles in biofluids, combined with their capacity to present cell-derived surface proteins, has led to growing interest in their potential as minimally invasive cancer biomarkers. While EV-based assays are not currently implemented for early cancer diagnosis, methodological challenges related to EV isolation, detection sensitivity and assay reproducibility remain key barriers to their clinical translation. The aim of this study was to comprehensively profile EV-associated surface markers relevant to lung cancer and to evaluate their robustness and detectability across different assay formats, isolation methods, and biological matrices. A multi-step analytical workflow was implemented using four non- small-cell lung cancer (NSCLC) cell lines (A549, NCI-H1975, NCI-H460 and NCI-H520). Cell-surface marker expression was first characterised by flow cytometry to establish a biological reference. EVs were subsequently isolated using three approaches: polyethylene glycol (PEG) precipitation, size-exclusion chromatography (Izon qEV1), and polymer-based precipitation (SubXTM), and further characterised by nanoparticle tracking analysis (NTA). EV surface marker detection was performed using antibody-based bead immunoprecipitation coupled to Luminex FLEXMAP 3D® assays in both single-plex and multiplex formats. Plasma spike-in experiments using tumour-derived EVs were performed to assess assay sensitivity in a clinically relevant matrix, and plasma samples from patients with metastatic lung cancer were analysed to explore translational feasibility. Distinct and biologically meaningful EV surface marker profiles were observed across lung cancer models. CD9 and CD63 were consistently detected across cell line-derived EVs, plasma spike-in experiments and patient plasma samples, with CD63 producing the strongest signal. Multiplex immunoprecipitation improved detection of several lower-abundance markers, including CD24, CD90, and CD274, compared with single-plex capture. Both Izon and SubXTM isolation methods enabled detection of tumour-derived EVs in plasma, however, Izon SEC produced a higher marker signal intensities across spike concentrations. In conclusion, this study establishes a robust technical framework for EV surface marker profiling in lung cancer and demonstrates the feasibility of immunoprecipitation-based bead assays for detecting biologically relevant EV markers. These findings support the potential of EV-associated surface proteins as candidates for future non-invasive lung cancer screening and early detection strategies. Description: M.Sc.(Melit.) 2026-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/147101 Title: Characterisation of two clinical mutations of Xanthine oxidoreductase related to hyperuricaemia Abstract: Xanthine oxidoreductase (XOR) is a molybdoflavin enzyme and a homodimer with a mass of 290 kDa. In mammals, XOR exists in two interconvertible forms: Xanthine Dehydrogenase (XDH) and Xanthine Oxidase (XO). XOR catalyses the final steps of purine catabolism, converting hypoxanthine to xanthine and subsequently to uric acid. These two reactions can generate reactive oxygen species (ROS), specifically hydrogen peroxide and superoxide anion radicals. XOR has been associated with various health conditions, including hyperuricemia, oxidative stress, and hypertension. ROS generated by XOR contribute to endothelial dysfunction, with increased ROS levels reacting with nitric oxide (NO) to form peroxynitrite radicals, exacerbating hypertension. Different variants have also been linked with increased XOR activity as well as higher risk of diseases, such as hyperuricemia and hypertension. In this project, the hXOR wildtype (WT) and two known clinical variants identified in a Maltese study by the MAMI study, I646V and I703V, as well as a novel double mutant (I646V-I703V) also identified in the Maltese population, were characterised. Apart from the clinical variants, the I646A mutation was also constructed to characterise to assess the effect of the substitution of a large non-polar branched amino acid such as isoleucine with a smaller one. The aim of this project was to determine the effect these amino acid changes have on the hXOR protein. Transformed TP1000 E.coli cells harbouring either the pTrc-HisXOR WT or the variants were grown under aerobic conditions without and with heat shock. Purification was performed using immobilised metal affinity chromatography. The purified proteins were characterised using SDS-PAGE, Native-PAGE, Blue-Native gel, and Dynamic Light Scattering (DLS) for size and oligomeric state. The enzymatic activity was evaluated using the uric acid spectrophotometric assay and the Nitro Blue Tetrazolium (NBT) assay. Secondary structure, thermal stability (melting temperature), and amyloid fibril formation were assessed using circular dichroism, DLS, and the Thioflavin T (ThT) assay, respectively. The amyloidogenic potential was further investigated using the ZipperDB server. All variants exhibited significantly higher enzymatic activities compared to the WT hXOR sample, with the I646A variant showing the highest activity (9-fold greater than WT). Analysis of DLS data confirmed that the native proteins of all mutants predominantly occur in the active native dimer conformation. Conversely, the WT hXOR exhibited the lowest activity and formed the largest oligomeric structures. The WT hXOR formed amyloid fibrils in the ThT assay, supported by the identification of amyloidogenic stretches using ZipperDB. Most variants did not show fibrillization, specifically I703V, I646A, I646V+HS, I646V-I703V+HS, and I646A+HS. The substitution of Ile646 with Valine or Alanine (I646V, I646A) significantly reduced the amyloidogenic propensity while increased substantially the enzymatic activity. I646V-I703V+HS also exhibited high enzymatic activity and displayed no fibril formation. These findings show that while WT hXOR activity is dampened by fibril formation, the clinical variants remain hyperactive and stable. This high activity of hXOR may result in significant ROS production and hyperuricemia potentially leading to various health conditions, such as hypertension. Description: M.Sc.(Melit.) 2026-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/146315 Title: MRI perfusion of solitary parotid lesions : a prospective study Abstract: Background: Parotid tumours (PTs) have a variety of pathological subtypes, each requiring distinct surgical approaches. Nevertheless, the current preoperative diagnostic methods often fall short in providing accurate PT diagnoses. Aim: Can DCE-MRI help predict histology in patients with focal parotid lesions? Design: A prospective study recruiting patients with known focal lesions of the parotid gland who are being worked up for surgery. A dynamic contrast-enhanced MRI (DCE-MRI) scan is performed, after which post-processing of data is used to obtain MR perfusion quantitative parameters (Ktrans, Kep and Ve) obtained by drawing a region of interest (ROI) over the PT. In this study 2 individual sets ROIs were drawn by two authors. The first set of ROIs vary in size, whereas the second are a fixed range ROI size limited to 10-20mm². The information obtained from DCE-MRI is compared to tissue histology and cytology, to assess the diagnostic accuracy of the DCE-MRI. The intra and inter-observer correlation of the different ROI methods is also assessed. Ethical standards were adhered to, with informed consent obtained from all participants. The data collection and analysis process spanned approximately 10 months. Methods: In this prospective study, 15 patients with histologically or cytologically proven PTs who underwent DCE-MRI were enrolled including 7 pleomorphic adenomas (PAs) and 8 Warthin’s tumours (WTs). Quantitative parameters of DCE-MRI (Ktrans, Kep, and Ve) of lesions were calculated and analysed. A second set of analysis was done to assess the intra and inter-observer correlation when using different ROI techniques. Results: Statistically significant (p < 0.05) inter-group differences were found between PA and WT for Kep and Ve. Correlation with histological results using the Mann-Whitney U test, where Ktrans, Kep and Ve were found to have p-values of 0.132, 0.008 and 0.003 respectively. Inter-observer correlation for the first set of variable size ROIs for the different MRI parameters were: Ktrans 0.590, Kep 0.622 and Ve 0.848, and for the second fixed range set Ktrans 0.945, Kep 0.901 and Ve 0.964. The intra-observer results for author 1 for the different MRI parameters were: Ktrans 0.358, Kep 0.718 and Ve 0.436. The intra-observer results for author 2: Ktrans 0.950, Kep 0.581 and Ve 0.762. Conclusions: In summary, DCE-MRI perfusion parameters aid in the differentiation of PA and WT. This technique demonstrates reliability and reproducibility. Standardised ROI sizes with fixed ranges minimise the impact of radiologist experience on perfusion parameter calculations, resulting in better interobserver agreement. However, when evaluating the reproducibility of perfusion parameters obtained by the same reader using different ROI techniques, radiologist experience significantly affects parameter calculations. Description: M.Sc.(Melit.) 2024-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/146314 Title: The use of shear-wave elastography in differentiating renal cell carcinoma and renal oncocytoma Abstract: Background: Renal tumours pose a significant diagnostic challenge, particularly in distinguishing malignant renal cell carcinomas (RCCs) from benign renal oncocytomas (ROs). Aim: This study aimed to evaluate the efficacy of shear wave elastography (SWE) in characterising RCCs and ROs based on tissue stiffness. Methods: Conducted at Mater Dei Hospital, Malta, from September to November 2024, this prospective study included 33 participants presenting 36 renal lesions. SWE was performed using a Canon Aplio a550 ultrasound system, and quantitative elasticity metrics were analysed to differentiate between RCCs and ROs. Results: Mann-Whitney U tests proved no statistical significant difference between RCCs and ROs. Mean speed measured for ROs was 2.43m/s ± 0.72m/s and 2.87m/s ± 0.83m/s for RCCs. Area under the curve (AUC) in receiver operator characteristic (ROC) curve analysis mean speed m/s was 0.660. SWE demonstrated no reliability in differentiating between RCCs and ROs. Conclusion: The results highlight the need for research into this field and emphasise the need for standardisation of methodology and development and further study of SWE in differentiating renal tumours with greater precision and confidence. Description: M.Sc.(Melit.) 2025-01-01T00:00:00Z